Method and quantification assay for determining c-kit/SCF/pAKT status

ABSTRACT

This invention provides methods for determining or predicting response to cancer therapy in an individual using differential image analysis of immunohistochemically stained tumor samples.

This application is a continuation of U.S. application Ser. No.10/225,826, filed Aug. 21, 2002 which claims priority to U.S.provisional application Ser. No. 60/314,188, filed Aug. 21, 2001.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for determining or predicting responseto cancer therapy in an individual. The invention also relates tomethods for using image analysis of immunohistochemically-stained tissuebiopsies to quantify c-kit/SCF/pAKT expression and activation, and toidentify efficacious anticancer compounds.

2. Background of the Invention

A primary goal of cancer therapy is to selectively kill or inhibituncontrolled growth of malignant cells while not adversely affectingnormal cells. Traditional chemotherapeutic drugs are highly cytotoxicagents that preferably have greater affinity for malignant cells thanfor normal cells, or at least preferentially affect malignant cellsbased on their high rate of cell growth and metabolic activity. However,these agents often harm normal cells.

Generally, anticancer drugs, monoclonal antibodies, chemotherapeutic orchemopreventive agents are used to produce growth arrest, terminaldifferentiation and cell death of the cancerous or precancerous cells(Mendelsohn, 1990, Semin. Cancer Biol. 1:339-44; Hancock et al., 1991,Cancer Res. 51:4575-80; Arteaga et al., 1994, Cancer Res., 54:3758-65;Pietras et al., 1994, Oncogene 9:1829-38; Bacus et al., 1997, Anal.Quant. Cytol. Histol. 19:316-28; Bacus et al., 1999, Breast J; Baselgaet al., 1999, Proceedings of AACR NCI EORTC International Conference,Abstract 98; Cobleigh et al., 1999, J. Clin. Oncol. 17:2639-48;DiGiovanna, 1999, PPO Updates: Princ. Practice Oncol. 13:1-9;Hortobagyi, 1999, J. Clin. Oncol. 17:25-29; Shak, 1999, Semin. Oncol.26:71-77; Sliwkowski et al., 1999, Semin. Oncol. 26:60-70; Vincent etal., 2000, Cancer Chemother. Pharmacol. 45: 231-38). Drug-induced growtharrest or cell death is often characterized by morphological andbiochemical changes associated with programmed cell death or terminaldifferentiation (as opposed to mitotic cell death).

Although chemotherapeutic drugs can be administered at doses high enoughto bring about cell death, such doses typically produce deleteriouseffects on normal as well as tumor cells. Differentiating agents, andthe use of lower doses of chemotherapeutic drugs and agents frequentlyresults in growth arrest rather than cell death. Such growth arrest canbe followed by apoptosis and cell death, or continued proliferation oncethe chemotherapeutic drugs are withdrawn. Administration of cytotoxicand chemotherapeutic drugs or ionizing radiation may also inducetransient growth arrest, a state that depends largely on the function ofp53 and p53-regulated cyclin-dependent kinase inhibitors (such as p16,p27, and p19) or growth inhibitors (such as TGF-β, IL-4, and IL-6). Uponremoval of the chemotherapeutic drug, cells subjected to the drugtreatment will eventually resume division and either continue toproliferate or die. Some drug-treated tumor cells undergo prolongedgrowth arrest and fail to resume cell division upon release from thedrug.

Apoptosis is generally regarded as an active suicide response to variousphysiological or pathological stimuli. Recent studies have shown that avariety of DNA-damaging agents, including X-ray irradiation and severalchemotherapeutic drugs (e.g. alkylating agents and topoisomerase IIinhibitors) initiate pathways leading to apoptosis. The exact mechanismby which apoptosis is induced by these agents is not yet known. However,expression of the tumor suppressor gene p53 has been implicated in thisprocess (Kwok and Sutherland, 1989, J. Natl. Cancer Inst. 81:1020-24;Kwok and Sutherland, 1991, Int. J. Cancer 49:73-76; Lane, 1992, Nature358:15-16; Kuerbitz et al., 1992, Proc. Natl. Acad. Sci. U.S.A.89:7491-95; Luo et al., 1995, Nature 375:159-61; Liu et al., 1996,Cancer Res. 56:31-35; Mellinghoff and Sawyers, 2000, PPO Updates 14:1-11). In addition, the up-regulation of caspases (e.g., caspase 9 orcaspase 3) or their chaperone molecules (e.g., heat shock protein 60)has been associated with apoptosis.

Cells can be rendered resistant to apoptosis in a variety of ways,including deletion of cellular genes such as the tumor suppressor genePTEN, overexpression of active Ras, and overexpression of active PI3K. Aparticular cellular protein, AKT (the protein product of the c-akt gene)has been identified as a key regulator of cellular survival and aninhibitor of apoptosis, which has significant implications foroncogenesis and drug resistance. For example, the loss of PTENcorrelates with increased AKT activity (Li et al., 1997, Science275:1943-47; Liaw et al., 1997, Nat. Genet. 16:64-67; Nelen et al.,1997, Hum. Mol. Genet. 6:1383-87; Cantley and Neel, 1999, Proc. Natl.Acad. Sci. U.S.A. 96:4240-45; Datta et al., 1999, Genes Dev.13:2905-27). In addition, suppression of apoptosis is not the onlyfunction that AKT may have in promoting oncogenesis. In somecircumstances, AKT can also induce cell cycle progression. However, theobservation that AKT can suppress apoptosis suggests that oncogenes mayblock adaptive cellular apoptosis by hyperactivating AKT.

Given the complexity of the apoptotic machinery, there are a number ofpathways by which AKT might act to promote cell survival and inhibitcell death. AKT may block apoptosis by regulating expression or activityof members of Bcl-2 gene family (that are known to play a role in cellsurvival or cell death). Alternatively, AKT may regulate expression oractivity of the caspase family of proteins, or the function of deathreceptor pathways. The regulatory effect of AKT may be through a directmechanism—the phosphorylation of components of the apoptotic machinery,for example—or an indirect mechanism—such as by altering the expressionlevel of genes that encode components of the death machinery. Recentstudies suggest that AKT regulates apoptosis at multiple sites. A numberof AKT targets, all playing critical roles in mediating cell death, havebeen identified, including BAD, caspase-9, the Forkhead family oftranscription factors, and the NFκB regulator IKK (Datta et al., 1999,supra).

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase growthfactor receptor that is placed in the same class as the receptors forPDGF and CSF-1 by virtue of their similar immunoglobulin-likeextracellular domains and the interruptions of their cytoplasmictyrosine kinase domain by a hydrophilic insert. (Yarden et al., 1987,EMBO J. 6:3341-3351). Its ligand, known alternatively as stem cellgrowth factor (“SCF”), mast cell growth factor, kit ligand, or steelfactor, is an early hematopoietic growth factor that, in conjunctionwith other growth factors, supports the proliferation anddifferentiation of multiple hematopoietic lineages. Id.

In several small-cell lung cancer cell lines and breast cancer celllines, it has been demonstrated that co-expression of c-kit and SCFoccurs, suggesting that autocrine growth stimulation may play a role innon-hematopoietic tumors. Autocrine growth requires co-expression of SCFand c-kit. In two independent studies, immunostaining of frozen sectionsusing polyclonal antisera directed against the carboxyl terminus ofc-kit has demonstrated uniformly strong staining of normal breast ductalepithelial cells. These studies also demonstrated that, using the samemethodology, at least 10-20% of breast carcinomas retain c-kitexpression. Neither of these studies, however, addressed the expressionof SCF in breast tumors. It is possible, therefore, that theco-expression of c-kit and SCF could play a role in the growthregulation of some breast tumors. (Hines, et al., 1995, Cell Growth &Differentiation, 6:769-779; Natali et al., 1992, Int. J. Cancer,52:713-717; Chui et al, 1996, British J. of Cancer, 73:1233-1236). Inaddition, co-expression of c-kit and other growth factors in breastcancer results in enhanced sensitivity to the EGF family of growthfactors. (Hines et al., 1999, Breast Cancer Research and Treatment 58:1-10).

Therefore, there exists a need in the art for improved detection ofexpression and activation of proteins and ligands that are directly orindirectly responsible for the expression or activation of AKT. There isalso a need for improved detection of expression and activation of c-kitand expression of its ligand SCF.

SUMMARY OF THE INVENTION

This invention provides reagents and methods for identifying anddetecting expression or activation of biological markers oftumorigenesis in cells and tissue samples from cancer patients. Themethods provided herein are useful for predicting or assessing aresponse of an individual cancer patient to a particular treatmentregimen, and methods useful for identifying efficacious anticancercompounds.

In a first aspect, the invention provides methods for assessing aresponse to administration of a chemotherapeutic or chemopreventiveagent to an individual, comprising:

-   -   (a) obtaining a first tissue or cell sample from the individual        before exposing the individual to the chemotherapeutic or        biological therapeutic agent;    -   (b) obtaining a second tissue or cell sample from the individual        after exposing the individual to the chemotherapeutic or        biological therapeutic agent;    -   (c) detecting the amount of one or a plurality of biological        markers in the first tissue or cell sample and in the second        tissue or cell sample;    -   (d) comparing the amount of the one or a plurality of biological        markers in said first tissue or cell sample with the amount in        said second tissue or cell sample;    -   (e) determining whether expression or activation of the one or a        plurality of biological markers decreases following exposure to        the chemotherapeutic or biological therapeutic agent when the        amount of the one or a plurality of biological markers in the        second tissue sample is less than the amount of the one or a        plurality of biological markers in the first tissue sample;        wherein a response to the administration of a chemotherapeutic        or biological therapeutic agent to an individual is assessed.

In certain embodiments, the one or a plurality of biological markers areexpressed from tumor related genes. In other embodiments, the one or aplurality biological markers are cellular components of a tumor-relatedsignaling pathway. In yet other embodiments, the biological markers arec-kit, SCF, pAKT, or pc-kit. In certain embodiments, the method ofdetecting the amount of the one or a plurality of biological markerscomprises using an antibody or antibodies that are immunologicallyspecific for the one or a plurality of biological markers. In otherembodiments, the amount of one or a plurality of biological markers aredetermined immunohistochemically.

In yet other embodiments, the method of detecting the amount of one or aplurality of biological markers comprises,

-   -   (a) staining the first and second tissue or cell samples using        one or a plurality of antibodies detectably-labeled with an        optical density label, wherein at least one antibody is        immunologically specific for one or a plurality of biological        markers, wherein the biological markers are c-kit, SCF, pAKT, or        pc-kit; and    -   (b) determining an optical density of the stained first and        second tissue sample in subpart (a);        wherein said optical density corresponds to the amount of said        one or a plurality of biological markers.

In other embodiments, the optical density is determined using imageanalysis. In further embodiments, the detectable label is a chromagen ora fluorophore. In yet further embodiments, the detectable label is DAB.In other embodiments, the agent is Glivac™.

In a second aspect, the invention provides methods for predicting theresponse to administration of a chemotherapeutic or chemopreventiveagent to an individual, comprising:

-   -   (a) obtaining a tissue or cell sample from the individual before        exposing the individual to the chemotherapeutic or biological        therapeutic agent;    -   (b) detecting the amount of one or a plurality of biological        markers in the tissue or cell sample;    -   (c) comparing the amount of one or a plurality of biological        markers determined in subpart (b) with the amount of one or a        plurality of samples expressing known amounts of the markers in        one or a plurality of control samples; and    -   (e) determining the expression or activation level of the one or        a plurality of biological markers compared with the expression        or activation level markers in one or a plurality of control        samples;        wherein a response to the administration of a chemotherapeutic        or biological therapeutic agent to an individual is predicted        based on the determination of decreased expression or activation        level of the one or a plurality of biological markers.

In certain embodiments, the one or a plurality of biological markers areexpressed from tumor related genes. In other embodiments, the one or aplurality biological markers are cellular components of a tumor-relatedsignaling pathway. In yet other embodiments, the biological markers arec-kit, SCF, pAKT, or pc-kit. In certain embodiments, the method ofdetecting the amount of the one or a plurality of biological markerscomprises using an antibody or antibodies that are immunologicallyspecific for the one or a plurality of biological markers. In otherembodiments, the amount of one or a plurality of biological markers aredetermined immunohistochemically.

In yet other embodiments, the method of detecting the amount of one or aplurality of biological markers comprises,

-   -   (a) staining the first and second tissue or cell samples using        one or a plurality of antibodies detectably-labeled with an        optical density label, wherein at least one antibody is        immunologically specific for one or a plurality of biological        markers, wherein the biological markers are c-kit, SCF, pAKT, or        pc-kit; and    -   (b) determining an optical density of the stained first and        second tissue sample in subpart (a);        wherein said optical density corresponds to the amount of said        one or a plurality of biological markers.

In other embodiments, the optical density is determined using imageanalysis. In further embodiments, the detectable label is a chromagen ora fluorophore. In yet further embodiments, the detectable label is DAB.In other embodiments, the agent is Glivac™.

In a third aspect, the invention provides methods for identifying acompound as a chemotherapeutic or biological therapeutic agent,comprising:

-   -   (a) obtaining a first tissue or cell sample;    -   (b) obtaining a second tissue or cell sample;    -   (c) exposing the second tissue or cell sample to said compound;    -   (d) detecting the amount of one or a plurality of biological        markers in the first tissue or cell sample and the second tissue        or cell sample;    -   (e) comparing the amount of the one or a plurality of biological        markers in said first tissue or cell sample and second tissue or        cell sample;    -   (d) (f) determining whether expression or activation of the one        or a plurality of biological markers decreases following        exposure to said compound when the amount of the one or more        biological markers in the second tissue or cell sample is less        than the amount of the one or a plurality of biological markers        in the first tissue or cell sample;        wherein the compound is identified as a chemotherapeutic or        biological therapeutic agent.

In certain embodiments, the one or a plurality of biological markers areexpressed from tumor related genes. In other embodiments, the one or aplurality biological markers are cellular components of a tumor-relatedsignaling pathway. In yet other embodiments, the biological markers arec-kit, SCF, pAKT, or pc-kit. In certain embodiments, the method ofdetecting the amount of the one or a plurality of biological markerscomprises using an antibody or antibodies that are immunologicallyspecific for the one or a plurality of biological markers. In otherembodiments, the amount of one or a plurality of biological markers aredetermined immunohistochemically.

In yet other embodiments, the method of detecting the amount of one or aplurality of biological markers comprises,

-   -   (a) staining the first and second tissue or cell samples using        one or a plurality of antibodies detectably-labeled with an        optical density label, wherein at least one antibody is        immunologically specific for one or a plurality of biological        markers, wherein the biological markers are c-kit, SCF, pAKT, or        pc-kit; and    -   (b) determining an optical density of the stained first and        second tissue sample in subpart (a);        wherein said optical density corresponds to the amount of said        one or a plurality of biological markers.

In other embodiments, the optical density is determined using imageanalysis. In further embodiments, the detectable label is a chromagen ora fluorophore. In yet further embodiments, the detectable label is DAB.

This invention provides reagents and methods for detecting expressionand activation of proteins and ligands that are directly responsible forinducing c-akt gene expression and/or activating AKT protein. Disclosedherein is the discovery that the c-kit proto-oncogene and its ligand,stem cell growth factor (“SCF” or “SCGF”) are co-expressed in variousamounts in breast cancers and that c-kit activation results inproduction of phosphorylated AKT (pAKT).

The invention also provides diagnostic methods for detecting andmeasuring c-kit, SCF, pc-kit and pAKT expression and protein levels anddetecting and measuring their activation states for the treatment oftumors. The invention provides reliable assays for determining c-kit,SCF, pc-kit and pAKT protein levels and their activation state in cellsor tissue samples obtained from patients that also permits a pathologistto exclude normal tissue from the analysis. Moreover, the inventionprovides methods for determining whether a cancer patient will benefitfrom a course of treatment using therapeutic agents directed atcomponents of the c-kit signaling pathways. The invention furtherprovides methods for monitoring a course of treatment using therapeuticagents directed at components of the c-kit signaling pathway. Theinvention also provides therapeutic agents identified by the inventivemethods.

Specific embodiments of the present invention will become evident fromthe following more detailed description of certain preferred embodimentsand the claims.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1D illustrate an immunohistochemical analysis of ahuman breast tissue sample identified by No. 12669b9. FIG. 1A shows aphotomicrograph of the sample tissue immunohistochemically stained withan antibody immunohistochemically-specific for SCF, as described inExample 2, at 400× magnification using a technique described in Example5. FIG. 1B shows the same breast cancer sample stained with an antibodyto c-kit (400×), as described in Example 1. FIG. 1C shows a differentfield of the same cancer tissue stained for SCF (400×). FIG. 1D showsanother field stained for c-kit (400×).

FIGS. 2A through 2E show the results of immunohistochemical analyses oftumor samples. FIG. 2A is a tumor stained for pAKT. FIG. 2B show tumorsample 747 stained for c-kit. FIG. 2C shows sample 848 stained for SCF.FIGS. 2D and 2E are different fields of tumor sample 920 stained foreither c-kit (FIG. 2D) or SCF (FIG. 2E).

FIG. 3 is a bar graph showing the levels of c-kit, SCF, and pAKTmeasured by immunohistochemistry, combined with image analysis. The bargraph is expressed in units of optical density/pixel.

FIG. 4 is photograph of a western blot showing c-kit activation inbreast cancer cells treated with SCF ligand in the presence of absenceof STI-571 (Glivac, obtained from Novartis Pharmaceuticals). pc-kit wasimmunoprecipitated from MDA-MB-361 cell lysates (50 μg total protein),separated by gel electrophoresis and screened with anti-phosphotyrosineantibodies to detect active c-kit receptor. As expected, the c-kitreceptor was activated by SCF treatment, but inhibited with as little as1 μM STI-571.

FIG. 5 is a photograph of a western blot of c-kit prepared from HeLacells. Rabbit polyclonal antibodies against c-kit were obtained fromNeoMarkers (1:200 dilution) and DAKO (1:400 dilution), and detectedusing HPR-conjugated antibodies against rabbit IgG (Amersham) andchemiluminescent substrate (NEN).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention provides methods for quantitatively determiningexpression and activation levels of tumor-related genes. The inventionspecifically provides methods for quantitatively determining expressionand activation levels for cellular proteins encoded by c-kit, SCF andAKT, in tumor cells, including human tumor cells, as detected in cell ortissue samples from an individual.

c-kit and its ligand SCF are co-expressed in various amounts in breastand bladder cancers, and the downstream signal associated with the c-kitactivation by SCF is phosphorylation of substrate AKT to phosphorylatedAKT (pAKT). According to the invention, expression and activation ofc-kit, SCF, and AKT are analyzed and monitored to identify patients thatcan benefit from treatment with a chemotherapeutic or biologicaltherapeutic agent, to monitor patients treated with such agents, or toidentify chemotherapeutic or biological therapeutic agents. For example,quantitative image analysis together with immunohistochemistry are usedaccording to the invention to analyze expression of these three tumormarkers, in order to identify patients that can benefit from treatmentwith a chemotherapeutic or biological therapeutic agent, including butnot limited to tyrosine kinase inhibitors. In particular embodiments,such tyrosine kinase inhibitors include but are not limited to STI-571,which is known to inhibit c-kit kinase. (Drucker, 2001, Am. Soc. OfClinical Oncology, 2001 Educational Book 37^(th) Annual Meeting, SanFrancisco, Calif.).

In contrast to traditional anticancer methods, where chemotherapeuticdrug treatment is undertaken as an adjunct to and after surgicalintervention, neoadjuvant (or primary) chemotherapy consists ofadministering drugs as an initial treatment in cancer patients. Oneadvantage of such an approach is that, for primary tumors of more than 3cm, it permits the use of conservative surgical procedures (as opposedto, e.g. radical mastectomy in breast cancer patients) for the majorityof patients. Another advantage is that for many cancers, a partialand/or complete response is achieved in about two-thirds of all cases.Finally, because the majority of patients are responsive after two tothree cycles of chemotherapeutic treatment, it is possible to monitorthe in vivo efficacy of the chemotherapeutic regimen employed, which isimportant for a timely identification of those cancers which arenon-responsive to chemotherapeutic treatment. Timely identification ofnon-responsive tumors, in turn, allows the clinician to limit the cancerpatient's exposure to unnecessary side-effects of treatment and toinstitute alternative treatments. However, the methods present in theart, including histological examination, are insufficient for such atimely and accurate identification. The present invention providesmethods by which a more informed and effective region of therapy can beadministered. In addition, the present invention provides methods bywhich the responsiveness or non-responsiveness of a therapy regimen canbe assessed, thereby limiting the patient's exposure to thechemotherapeutic drug.

A cancer diagnosis, both initial diagnosis of disease and subsequentmonitoring of the disease course, before, during, or after treatment, isconventionally confirmed through histological examination of cell ortissue samples removed from a patient. Clinical pathologists need to beable to accurately determine whether such samples are benign ormalignant and to classify the aggressiveness of tumor samples deemed tobe malignant, because these determinations often form the basis forselecting a suitable course of patient treatment. Similarly, thepathologist needs to be able to detect the extent to which a cancer hasgrown or gone into remission, particularly as a result of or consequentto treatment, most particularly treatment with chemotherapeutic orbiological agents.

Histological examination traditionally entails tissue-stainingprocedures that permit morphological features of a sample to be readilyobserved under a light microscope. A pathologist, after examining thestained sample, typically makes a qualitative determination of whetherthe tumor sample is malignant. It is difficult, however, to ascertain atumor's aggressiveness merely through histological examination of thesample, because a tumor's aggressiveness is often a result of thebiochemistry of the cells within the tumor, such as protein expressionor suppression and protein activation, which may or may not be reflectedby the morphology of the sample. Therefore, it is important to be ableto assess the biochemistry of the cells within a tumor sample. Further,it is desirable to be able to observe and quantitate both geneexpression and protein activation of tumor related genes or proteins, ormore specifically cellular components of a tumor-related signallypathway.

Automated (computer-aided) image analysis systems known in the art canaugment visual examination of samples. In a representative system, thecell or tissue sample is exposed to detectably labeled reagents specificfor a particular biological marker, and the magnified image of the cellis then processed by a computer that receives the image from acharge-coupled device (CCD) or camera such as a television camera. Sucha system can be used, for example, to detect and measure expression andactivation levels of the c-kit, SCF, pc-kit, and pAKT in a sample. Thismethodology provides more accurate diagnosis of cancer and a bettercharacterization of gene expression in histologically identified cancercells, most particularly with regard to expression of tumor marker genesor genes known to be expressed in particular cancer types and subtypes(i.e., different degrees of malignancy). This information permits a moreinformed and effective regimen of therapy to be administered, becausedrugs with clinical efficacy for certain tumor types or subtypes can beadministered to patients whose cells are so identified. A non-limitingexample is administration of tyrosine kinase inhibitors to patientshaving tumor biopsy or other test samples that test positive for c-kit,SCF, pc-kit, and pAKT.

Another drawback of conventional anticancer therapies is that theefficacy of specific chemotherapeutic agents in treating a particularcancer in an individual human patient is unpredictable. In view of thisunpredictability, the art is unable to determine, prior to startingtherapy, whether one or more selected agents would be active asanti-tumor agents or to render an accurate prognosis of course oftreatment in an individual patient. This is especially important becausethe same clinical cancer may present the clinician with a choice oftreatment regimens, without any current way of assessing which regimenwill be most efficacious for a particular individual. It is an advantageof the methods of this invention that they are able to better assess theexpected efficacy of a proposed therapeutic agent (or combination ofagents) in an individual patient. Additional features of the claimedmethods for assessing the efficacy of chemotherapeutic regimens are thatthey are both time- and cost-effective and minimally traumatic to cancerpatients.

In the practice of one embodiment of the methods of this invention, atwo-component immunohistochemical staining system is used. In thisembodiment, cell pellets or tissues are counterstained with a dye, stainor detectable label producing a first color while proteins of interestin the cell pellet or tissue sample are stained with a dye, stain ordetectable label producing a second, different color. The image of thecells in the cell pellet and tissue sample is then magnified in a lightmicroscope and split into a pair of separated images. A pair of opticalfilters that are specifically matched to have a maximum absorption foreach specific stain is used to enhance the separated images. One of theoptical filters preferentially transmits light having a wavelength atthe absorption wavelength of the counterstained tissue. The other,preferably narrow bandpass optical filter preferentially transmits lightin the regions of spectral absorption for the dye, stain or detectablelabel used to detect the protein of interest. Using image analysisfilters, different cellular proteins in various components, such as themembrane, cytoplasm and nucleus, can be quantified. For optimal results,the imaging system is calibrated prior to taking any measurements.

One of the advantages of the use of methods of this invention thatutilize two-component immunohistochemical staining is that thesubcellular localization of a protein of interest can be determined.

In the practice of another embodiment of the methods of the invention,the amount of target protein in a biological sample is quantitated(i.e., the amount of target protein is measured) using calibrationcurves that relate the amount and degree of target protein-specificstaining to the amount of the target protein in the sample. This is mostgenerally accomplished by comparing experimental or patient samples withcell samples prepared using cells, most preferably cultured cell linesthat produce a consistent amount of the target protein that can bedetermined with high degrees of accuracy and precision. In certainpreferred embodiments, a plurality of cell populations are assayed thateach express different amounts of the target protein, to permit acalibration curve to be produced relating staining intensity with amountof protein in a sample. Such cell populations are used to determine theamount of target protein-specific staining associated with varyingamounts of target protein in the different cell populations. In certainpreferred embodiments the amount of target protein-specific staining isnormalized or expressed relative to the amount of total cell protein, toprovide a convenient measure for analyzing biological tissues,preferably tumor tissue and most preferably malignant tumor tissue, inwhich a determination of cell number would be impractical or unreliable.In the practice of the invention, the correlation between targetprotein-specific staining and the amount of target protein expressed ina cell is expressed as a calibration curve relating the amount of targetprotein to a physical parameter, most preferably optical density,associated with target protein-specific staining. The calibration curvesproduced according to and used with the methods of the invention arealso advantageously expressed as an algorithm, most preferably in theform of a linear or logarithmic equation, and are preferably automated,for example, by programming a device such as a computer to convert asample staining intensity to an amount of a target protein in thesample.

In the practice of another embodiment of the methods of the invention,the subcellular localization of a protein of interest can be determined.This is advantageous because some proteins, such as the estrogenreceptor, are localized in the cytoplasm of the cell until they areactivated, at which point they translocate into the nucleus where theycan participate in, for example, gene expression.

A description of exemplary procedures for quantitativeimmunohistochemical image analysis of tissue samples can be found,generally, in co-pending U.S. patent application Ser. Nos. 09/760,120and 09/760,121, both filed Jan. 12, 2001, and WO 01/51924 and WO01/51928, both filed Jan. 12, 2001, all of which are incorporated hereinby reference in their entirety. As an example, Quantitative imageanalysis was performed on a Cell Analysis Systems Model 200, availablefrom Becton Dickinson Company, Mountain View, Calif. Quantitative imageanalysis, as encompassed by this invention, can be performed on systemsfrom other vendors, such as, but not limited to, ChromaVision MedicalSystems (San Juan Capistrano, Calif.).

In the practice of the methods of this invention, target proteinsincluding but not limited to c-kit, SCF, pc-kit, and pAKT can bedetected using a specific reagent, most preferably an antibody, that isitself detectably labeled, or using an unlabeled, targetprotein-specific antibody and a second antibody that is detectablylabeled and recognizes the target protein-specific antibody.Alternatively, any molecule that can be detectably labeled and thatspecifically binds to the target protein can be used in the practice ofthe methods of the invention. In a preferred embodiment of the methodsof the present invention, a two-component immunohistochemical stainingsystem is used to differentially stain the target protein and the tissueor cell sample so that the stained target protein can be more readilydistinguished from the counterstained tissue or cell sample. Followingimmunohistochemical staining, the optical image of the tissue or cellsample preferably generated by a computer-aided image analysis system isthen magnified under a light microscope and separated into a pair ofimages. The separated images are enhanced using a pair of opticalfilters, one having a maximum absorption corresponding to the stain andthe other having a maximum absorption corresponding to the counterstain,thereby providing an optimum discrimination between the two stains. Inother embodiments of the method of the present invention, a plurality ofimage analysis filters are used to detect, differentiate, and quantitatestaining levels of different cellular proteins in various components(e.g., membrane, cytoplasm, and nucleus). In a non-limiting example, thetarget can be stained using diaminobenzidine (DAB) and the tissue orcell sample can be counterstained using ethyl green or methylene blue.

It will be recognized by those with skill in the immunohistochemistryarts that dye lot variations and variability in certain environmentalconditions, such as relative humidity can affect results obtained atdifferent times using the methods of the invention. In certainembodiments of the methods of the present invention, stain andcounterstain lot variability is controlled by using the same reagents tostain the sample cells and first and second control cell pellets. Inother certain embodiments of the methods of the present invention,environmental differences and variability in the staining procedure arecontrolled for by staining the sample cells and first and second controlcell pellets at the same time.

In certain embodiments, target protein-specific staining is detected,measured and quantitated using image analysis equipment, defined hereinas comprising a light or fluorescence microscope, an image-transmittingcamera and a view screen, most preferably also comprising a computerthat can be used to direct the operation of the device and also to storeand manipulate the information collected, most preferably in the form ofoptical density of certain regions of a stained tissue preparation.Image analysis devices useful in the practice of this invention includebut are not limited to the CAS 200 system (Becton Dickenson, MountainView, Calif.).

In certain embodiments, the inventive methods are practiced using suchan image analysis system as follows. After immunohistochemical stainingas described above, a quantified measure of the percentage of expressingcells can be taken by digitizing microscope images of stained samples,and converting light intensity values in each picture element (pixel) ofthe digitized image to optical density values, which correspond to thepercentage of stained cell components (such as nuclei). Morespecifically, computerized image analysis can be used to determine aquantity of cells having a particular stain using a digital grey scaleimage. Grey scale images are representative of the amount of an opticalenhancement factor, such as a chromagen, which binds to a specifictarget under study and thereby allows optical amplification andvisualization of the target.

As used in the practice of the methods of this invention, a computerizedimage analysis apparatus includes a means such as a lens for magnifyingand displaying the image of a group of cells of a specimen from a fieldon a microscope slide. The specimen cell population is prepared usingstaining and counterstaining techniques known in the immunohistochemicalarts, such as enzymatic staining methods and chemical staining methods.Appropriate stains and counterstains are selected by their ability toenable cells to be distinguished, inter alia, those that containantibody sandwich complexes comprising specific enzymes or markers andthose that do not contain such complexes, through cameras used with thecomputerized image analysis system. After staining, the image field isdigitized by the apparatus and stored in a memory provided by thesystem. From the digitized image, a nuclear or cytoplasmic image mask isformed by using one wavelength of light such as red wavelength or greenoptical filter for form the image. The tissue mask is stored and asecond filter is used to form another filtered image of the same areaswith the optical enhancement factor. Differentiation of cellularcharacteristics can be made by comparing the first image with the secondimage to obtain a quantification of material stained with the opticalenhancement factor and thus, an assay of the amount of the particulartarget under study.

In a first step, any expressed target protein in the cells is identifiedby adding a detectably-labeled primary antibody specific for the targetprotein, or alternatively an unlabeled primary antibody and adetectably-labeled secondary antibody specific for the primary antibody.The antibodies are incubated with the sample for a time to formcomplexes if these antigens are present. The complexes are thenvisualized, either by directly detecting the label, or, if thedetectable label is an enzyme, incubating the sections with a compound,such as a chromagen under appropriate conditions. As an example, theprimary antibody can be labeled peroxidase, with the subsequent use ofthe chromagen DAB. In a second step, the tissue is counterstained withanother optical enhancement factor, such as, but not limited to ethylgreen. Although a staining technique using peroxidase and ethyl green isexemplary, other stains and optical enhancement factors are alsosuitable. For example, other chromagens can be used with peroxidase,including, but not limited to 3-Amino-9-Ethylcarbazole (AEC), whichproduces a red color, and 4-Chloro-1-Naphthol. In addition, otherenzymes that can be used to label the primary antibody are encompassedby this invention, including, but not limited alkaline phosphatase.Examples of chromagens that can be used with alkaline phosphataseinclude, but are not limited to Fast Red, Fast Green, and5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT).In addition, the use of florescent dyes is encompassed by methods of thepresent invention. As non-limiting examples of florescent dyes, the useof Fluorescein (fluoroisothiocyanate or FITC) and Rhodamine.

Further, the use of ethyl green is exemplary of counter stains, but useof other counter stains are encompassed by the methods of the presentinvention. For example, a non-limiting list of additional counter stainsinclude hematoxylin, fast red, methyl green. A counter stain is chosenfor a particular stain based on the spectral separation that can beachieved such that filtering at two separate wavelengths can beachieved. For example, ethyl green offers good spectral separation fromthe DAB precipitate, as described in more detail below. Other examplesof pairs of stains/counter stains that offer good spectral separationinclude, but are not limited to, Fast Red and Hematoxylin, AEC andHematoxylin, and FITC and Texas Red. Those skilled in the art willrecognize that this list of stains and counter-stain is merely exemplaryand, therefore, does not serve to limit the invention.

As an example of the an embodiment of a method of the present invention,spectral studies have shown that the ethyl green stain offers goodspectral separation from the DAB precipitate produced byimmunoperoxidase and permits different features of the image to bereadily separated by filtering the image at two different wavelengths.This allows the image to be digitized into two separate images, forexample, one in which all the cell nuclei are optically enhanced (using,for example, ethyl green or Fast Green) and one in which only thosetissue areas with receptor staining (DAB) are optically enhanced. Theresulting tissue preparation stained with ethyl green will have greennuclei with varying degrees of brown DAB precipitate localized to thecytoplasm or nuclei, proportional to the level of expression of themarker protein. Although a staining technique using peroxidase and ethylgreen is described, other stains and optical enhancement factors arealso suitable (such as alkaline phosphatase coupled with a specificchromagen such as Fast Red or Fast Green counterstain). Spectral studieshave shown that the ethyl green stain offers good spectral separationfrom the DAB precipitate of the immunoperoxidase technique such thatfiltering at two different wavelengths can readily separate differentfeatures of the image. This allows the image to be digitized into twoseparate images, one in which all the cell nuclei are optically enhanced(ethyl green or Fast Green) and one in which only those tissue areaswith receptor staining (DAB) are optically enhanced. In a particularembodiment, the images can be separated by a 600 nanometer (red) filterto produce an image of the counterstained area and a 500 nanometer(green) filter to produce an image of the tissue areas staining with theDAB precipitate.

To further differentiate the stained areas, an interactive thresholdsetting technique can be used where an operator visualizing the imagescan set a boundary on the areas under consideration. When suchboundaries are set, images are formed by eliminating all parts of theimage that are below the thresholds in optical density. As practicedaccording to the teachings contained herein, a threshold isadvantageously set for the first image, and a second threshold is setfor the second image.

Further, the image processing method performed preferably using theimage analysis systems of the invention comprises first forming a maskimage of the tissues under consideration with, for example, a redfilter. This mask image is stored, preferably in the system's computermemory and another image for expressed protein quantification isacquired by using, for example, a green-filtered version of the sameimage. The effect of the filters in combination is to optically enhance(i.e., make darker) those areas of the tissue mask where tissuecomponents are stained with DAB and to make lighter those tissuecomponents stained only with the green counterstain. An image analysiscan then be performed using only those areas of the image that arestained and are within the mask.

The combination of red and green optical filters and DAB and ethyl greenor other green counterstains are advantageously used in the practice ofthe methods of the invention. This combination provides a convenient andadvantageous way to discriminate between two counterstained areas. Thosewith skill in the art will recognize that there are a plurality of otherstains, filters, staining or optical enhancement methods and filteringmethods that can be used to optically enhance one particular area orfeature over another cell feature, such as Fast green, eosin.

For example, expression and activation of proteins expressed fromtumor-related genes can be detected and quantitated using methods of thepresent invention. Further, expression and activation of proteins thatare cellular components of a tumor-related signaling pathway can bedetected and quantitated using methods of the present invention.Further, proteins associated with breast cancer can be quantified byimage analysis using a suitable primary antibody against Her-1, Her-2,p-Her-1, p-Her-2, p-ERK, c-kit, p-AKT, or SCF, or to theirphosphorylated forms, and a secondary antibody (such as rabbitanti-mouse IgG when using mouse primary antibodies) and/or a tertiaryavidin (or Strepavidin) biotin complex (“ABC”). Examples of reagentsinclude a rabbit polyclonal antibody specific for pAKT and obtained fromCell Signaling Technology (Beverly, Mass., Cat. No. 9277); anti-SCFantibody, obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.,Cat. No. SC-9132); and a polyclonal anti-c-kit antibody obtained fromNeomarkers, Inc. (Fremont, Calif., Cat. No. RB-1518).

In practicing the method of the present invention, staining procedurescan be carried out by a technician in the laboratory. Alternatively, thestaining procedures can be carried out using automated systems.

The amount of target protein can then be quantitated by the averageoptical density of the stained antigens. Also, the proportion orpercentage of total tissue area stained may be readily calculated, asthe area stained above an antibody threshold level in the second image.Following visualization of nuclei containing c-kit, SCF, and pAKT, thepercentage or amount of such cells in tissue derived from patients aftertreatment may be compared to the percentage or amount of such cells inuntreated tissue.

From these comparisons, the potential effectiveness of a putativetherapeutic or chemopreventive agent against a tumor can be determined.Specifically, if there is a greater amount of expression and activationof protein expressed from a tumor-related gene in the untreated portionof the sample than there is in the treated portion of the sample, thenthe therapeutic agent used in the treatment will be predicted to be aneffective agent for treating the cancer. Further, if there is a greateramount of expression and activation of protein that is a cellularcomponent of a tumor-related signaling pathway in the untreated portionof the sample than there is in the treated portion of the sample, thenthe therapeutic agent used in the treatment will be predicted to be aneffective agent for treating the cancer. Further, if there is a greateramount of expression and activation of c-kit, SCF, and pAKT in theuntreated portion of the sample than there is in the treated portion ofthe sample, then the therapeutic agent used in the treatment will bepredicted to be an effective agent for treating the cancer. Conversely,if there is a substantially equal percentage of cells expressing c-kit,SCF, and pAKT in both the treated and untreated portions of the sample,or there is less protein expression in the untreated portion of thesample, then the putative therapeutic agent will not be predicted to beeffective for treatment of the cancer. For example, this procedure canbe used to establish that a c-kit tyrosine kinase inhibitor, Novartisagent STI-571 (GLIVEC™), is predicted to be a potentially effectiveagent for treating a cancer sample.

Particularly useful embodiments of the present invention and theadvantages thereof can be understood by referring to Examples 1-7. TheseExamples are illustrative of specific embodiments of the invention, andvarious uses thereof. They are set forth for explanatory purposes only,and are not to be taken as limiting the invention.

Example 1 Staining Procedure for c-Kit

Staining for c-kit was performed on a Benchmark™ automated stainingmodule (Ventana Medical Systems, Inc., Tucson, Ariz.) using a polyclonalantibody to c-kit (NeoMarkers, Fremont, Calif.). 10% Neutral BufferedFormalin fixed 4 micron paraffin sections of the sample were place ontocoated slides. The Benchmark bar-code specific for the c-kit stainingprotocol was placed onto the slide and the slide placed onto theBenchmark. All subsequent steps, drying, deparaffinization antigenretrieval (Cell Conditioning 1 mM EDTA), and detection were performedautomatically by the Benchmark. All test reagents are available fromVentana Medical Systems, including the I-View DAB Detection Kit, unlessotherwise noted. The protocol implemented in using the automated systemis described in Table 1. Where indicated, one drop is one reagentdispense. The sample was then manually counterstained with 4% EthylGreen 0.1 M pH 4 acetate buffer for 10 minutes. The results of stainingfor c-kit are described below in Example 5.

TABLE I 1 ****Select EZ Prep**** 2 ****Start Timed Steps 3 ****MixersOff**** 4 Warmup Slide to 75° C., and Incubate for 4 minutes 5 ApplyEZPrep Volume Adjust 6 Incubate for 4 minutes 7 Rinse Slide + 8 ApplyEZPrep Volume Adjust 9 Incubate for 4 minutes 10 Rinse Slide + 11 ApplyEZPrep Volume Adjust 12 Apply Coverslip 13 Warmup Slide to 76° C., andIncubate for 4 minutes 14 Rinse Slide + 15 Apply Depar Volume Adjust 16Apply Coverslip 17 Warmup Slide to 42° C., and Incubate for 2 minutes 18****Mixers On**** 19 Rinse Slide + 20 Apply Medium Cell Conditioner #121 Apply CC Long Coverslip 22 Warmup Slide to 95° C., and Incubate for 8minutes 23 Apply Medium Cell Conditioner #1 24 Apply Coverslip 25 WarmupSlide to 100° C., and Incubate for 4 minutes 26 Apply Coverslip 27 ApplyCell Conditioner #1 28 Incubate for 4 minutes 29 Apply Coverslip 30Apply EZPrep CC Volume Adjust 31 Incubate for 4 minutes 32 ApplyCoverslip 33 Apply Medium Cell Conditioner #1 34 Incubate for 4 minutes35 Apply Coverslip 36 Apply Cell Conditioner #1 37 Incubate for 4minutes 38 Disable Slide Heater 39 Incubate for 8 minutes 40 RinseSlide + 41 Adjust Slide Volume 42 Apply Coverslip 43 ****Select ReactionBuffer**** 44 Warmup Slide to 42° C., and Incubate for 2 minutes 45Rinse Slide + 46 Adjust Slide Volume 47 Apply One Drop of I-VIEWINHIBITOR, Apply Coverslip, and Incubate 4 min. 48 Rinse Slide + 49Adjust Slide Volume 50 Apply One Drop of c-kit primary antibody [PREPKIT 4], Apply Coverslip, and Incubate 32 min. 51 Rinse Slide + 52 AdjustSlide Volume 53 Apply One Drop of I-VIEW BIOTIN Ig, Apply Coverslip, andIncubate 8 min. 54 Rinse Slide + 55 Adjust Slide Volume 56 Apply OneDrop of I-VIEW SA-HRP, Apply Coverslip, and Incubate 8 min. 57 RinseSlide + 58 Adjust Slide Volume 59 Apply Coverslip 60 Rinse Slide + 61Adjust Slide Volume 62 Apply One Drop of I-VIEW DAB and One Drop I-VIEWH₂O₂, Apply Coverslip, and Incubate 8 min. 63 Rinse Slide + 64 AdjustSlide Volume 65 Apply One Drop of I-VIEW COPEER, Apply Coverslip, andIncubate 4 min. 66 Rinse Slide +

Example 2 Staining Procedure for SCF

SCF staining was performed using a polyclonal antibody to SCF (SantaCruz Biotechnology, Santa Cruz, Calif.). A biotinylated anti-rabbitsecondary antibody (Jackson Research Labs, West Lake, Pa.). the“StreptABComplex-HRP” (Streptavidin/Biotin Complex with a HorseradishPeroxidase) label kit (DAKO Corporation, Carpinteria, Calif.), and theDAB chromagen (Dako) were used as the detection system for SCF. 10%Neutral Buffered Formalin fixed 4 micron paraffin sections of the samplewere place onto coated slides. To prepare the sample for staining, thesample was first deparaffinized and hydrated to water. Antigen retrievalwas preformed enzymatically with Digest-All 1 (Zymed Labs), a Ficindigestion solution. 1-2 drops of the manufacture's ready-to-use solutionwas added to the sections and incubated for 20 minutes at 37° C. Thesections were then washed well with deionized water and rinsed with TBS.Endogenous peroxidase was blocked by incubating the sections with a 3%hydrogen peroxide/methanol solution for 10 minutes, followed by adeionized water wash. The sections were then blocked using 10% blockinggoat serum in 0.1% BSA/0.1% Triton X for 10 minutes. The goat serum wasthen shaken off.

The sections were incubated with a 1:20 dilution of SCF primary antibodyat 37° C. for 45 minutes. The StreptABC label was prepared while theprimary antibody was incubating with the sections by adding 4 μls ofStreptavidin solution with 4 μls biotin solution in 1 ml of 50 mMTris-HCL buffer, pH 7.6. After SCF primary antibody incubation, thesections were washed well with TBS and incubated with a 1:300 dilutionof biotinylated Goat anti-rabbit IgG at 37° C. for 20 minutes. Thesections were washed well with TBS, followed by incubation with theStreptABC label at 37° C. for 20 minutes. The sections were washed wellwith TBS, and DAB liquid chromagen was applied at room temperature for 5minutes. The sections were then washed well with deionized water. Theslides were counterstained with 4% ethyl green. The results of stainingfor SCF are described below in Example 5.

Example 3 Staining Procedure for Phospho-AKT

p-AKT staining was performed using a Dako Autostainer with the LSAB2 kit(described above). 10% Neutral Buffered Formalin fixed 4 micron paraffinsections of the sample were place onto coated slides. To prepare thesample for staining, the sample was first deparaffinized and hydrated towater. Antigen retrieval was preformed with 0.1 M citrate retrievalbuffer, pH 6.0 in a Decloaker Chamber (Biocare Medical, Walnut Grove,Calif.) following the manufacturer's instructions. The sections wereallowed to cool for 15 minutes, and then washed well with deionizedwater. Endogenous peroxidase was blocked by incubating the sections witha 3% hydrogen peroxide/Methanol solution for 10 minutes, followed by adeionized water wash. The sections were then blocked using 10% blockinggoat serum in 0.1% BSA/0.1% Triton X for 10 minutes. The goat serum wasthen shaken off.

The sections were then incubated with a 1:75 dilution of p-AKT primaryantibody (Cell Signaling Technology, Beverly, Mass.) at 4° C. overnight.The next morning, the sections were removed from the refrigerator andallowed to come to room temperature, about 30 minutes. The slides werewashed well with TBS and loaded into the Dako Autostainer. TheAutostainer was programmed to apply the biotinylated link for 30minutes, the streptavidin-HRP reagent for 30 minutes, and the DABchromagen for 5 minutes. The slides were rinsed with TBS/Tween by theautostainer between the linking antibody, labeling, and DAB chromagenapplications. After DAB chromagen, the slides were removed from theautostainer and washed well with deionized water. The slides werecounterstained with 4% ethyl green. The results of staining for SCF aredescribed below in Example 5

Example 4 Staining Procedure for Phospho-KIT

pc-kit staining was performed using a Dako Autostainer with a reagentkit obtained from DAKO Corp. (the LSAB2 kit (labeledstreptavidin-biotin) containing a Horseradish Peroxidase Label). 10%Neutral Buffered Formalin fixed 4 micron paraffin sections of the samplewere place onto coated slides. To prepare the sample for staining, thesample was first deparaffinized and hydrated to water. Antigen retrievalwas preformed with 1 mM EDTA in a Decloaker Chamber (Biocare Medical,Walnut Grove, Calif.), an electric pressure cooker that can be utilizedfor heat-induced epitope retrieval (HIER) procedures, following themanufacturer's instructions. The sections were allowed to cool for 15minutes, and then washed well with deionized water. Endogenousperoxidase was blocked by incubating the sections with a 3% hydrogenperoxide/methanol solution for 10 minutes, followed by a deionized waterwash. The sections were then blocked using 10% blocking goat serum in0.1% BSA/0.1% Triton X for 10 minutes. The goat serum was then shakenoff.

The sections were then incubated with a 1:50 dilution of p-kit (Try 719)primary antibody (Cell Signaling Technology, Beverly, Mass.) at 4° C.overnight. The next morning, the sections were removed from therefrigerator and allowed to come to room temperature, about 30 minutes.The slides were washed well with TBS and loaded into the DakoAutostainer. The Autostainer was programmed to apply the biotinylatedlink for 30 minutes, the streptavidin-HRP reagent for 30 minutes, andthe DAB chromagen for 5 minutes. The slides were rinsed with TBS/Tweenby the autostainer between the linking antibody, labeling, and DABchromagen applications. After DAB chromagen, the slides were taken offthe autostainer and washed well with deionized water. The slides werecounterstained with 4% ethyl green. The result was the ability to detectthe level of expression or activation of pc-kit in the sample (data notshown).

Example 5 Immunohistochemical Staining of Tumor Sample for c-kit, SCF,and pAKT

To identify a patient to be a candidate for c-kit inhibitors, such asNovartis' Glivec™, tumor samples were examined for c-kit and SCFexpression. Paraffin blocks of tumor samples were sectioned at 4-5microns and the sections were placed onto coated slides. The slides werethen stained as described above for either c-kit, SCF, or pAKT. Theresults of these experiments are represented in FIGS. 1A through 1D.These Figures represent microphotographs of one individual's breastcancer sample. Two microphotographs (FIGS. 1A and 1C) represent fieldsstained with antibodies to SCF and two microphotographs (FIGS. 1B and1D) represent fields stained with antibodies to c-kit. All photographsshow staining and thus that the same tumor expresses both c-kit and SCF.Therefore, this breast cancer sample identified the patient as a goodcandidate for c-kit inhibitors.

As a further example of tumor samples that were determined to becandidates for c-kit inhibitors, FIG. 2A-2E represent either bladdercancer samples (2A, 2D, and 2E) or breast cancer samples (2B and 2C)stained with antibodies to c-kit, SCF, and pAKT, as described above.FIG. 2B represents breast tumor sample 747 stained for c-kit. FIG. 2Crepresents breast tumor sample 848 stained for SCF. FIGS. 2D and 2Erepresent bladder tumor sample 920 stained for both c-kit (FIG. 2D) andfor SCF (FIG. 2E), indicating that the patient is a good candidate forc-kit inhibitors. FIG. 2A represents the same bladder tumor sample 920stained for pAKT.

Example 6 Image Analysis of c-Kit, SCF, and pAKT Protein Expression

Tumor samples were stained with antibodies to c-kit, SCF, and pAKT, andimage analysis was used to determine the level of expression of thesethree proteins. The individual tumor samples analyzed included bothsamples of breast cancer (747, 848, 864, 895) and bladder cancer (920).The measurements were made using the CAS system as described in Bacus etal. (1997, Anal. Quant. Cytol. Histol. 19:316-28, incorporated herein byreference in its entirety). The levels of c-kit, SCF, and pAKT protein,expressed in units of optical density per pixel, are shown in Table II.FIG. 3 is a bar graph showing the levels of c-kit, SCF, and pAKTmeasured by image analysis and IHC staining in these five tumors showingthat all three proteins were expressed, that all three proteins could bemeasured, and that there was considerable variation in the expressionlevel of the three proteins. Presumably, these variations will affectdrug susceptibility of the cancer.

TABLE II Image analysis quantification of c-kit, pAKT, and SCF in TumorSample, Expressed as Optical Density per Pixel Sample c-kit PAKT SCF“747” 0.3 0.15 0.07 “848” 0.32 0.13 0.1 “864” 0.14 0.06 0.25 “895” 0.130.04 0.1 “920” 0.29 0.4 0.07

Example 7 Response to STI-571 in Breast Cancer Cell Lines

The breast cancer cell line MDA-MB-361 was used to determine theresponse of a breast cancer line to a c-kit tyrosine kinase inhibitor,specifically Novartis agent STI-571 (GLIVEC™). FIG. 4 is arepresentation of a western blot showing c-kit activity in breast cancercells treated with SCF ligand in the presence or absence of STI-571 incell culture. The cell line was exposed to STI-571, varying from 1 μM to40 μM, during a three-day cell culture. The cultured cell line and thecontrol, untreated cell line were then lysed and cell lysates with 50 μgtotal protein were immunoprecipitated with both the NeomarkersCD117/c-Kit antibody clone Ab-6, and the DAKO CD117/c-Kit antibody cloneA4502. FIG. 5 is a representation of a western blot of c-kit expressionin HeLa cells probed with the same antibodies used to immunoprecipitatec-kit in the present experiment, clearly showing that both of theantibodies were capable of isolating c-Kit. FIG. 4, lane 1 shows thatthere is no activated c-kit expressed in the absence of its ligand, SCF.Lane 2 shows a distinct band at 145 kDa that indicates the isolation ofactivated c-kit when the cell culture is exposed to SCF. The remaininglanes (3-6) do not have a band at 145 kDa, demonstrating that there isno c-kit expression in the presence of as little as 1 μM STI-571. Thisresult shows that STI-571 (GLIVEC™) is capable decreasing the amount ofexpression or activation of c-kit, in the presence of its ligand, SCF.Therefore, STI-571 (GLIVEC™) is identified as a compound that is usefulto decrease expression or activation of c-kit, and correspondinglydecrease activation of AKT.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

What I claim is:
 1. A method comprising: immunohistochemically staininga first breast tissue or first breast cell sample from a breast cancerpatient to whom a c-kit inhibitor has been administered with one or aplurality of detectably-labeled antibodies, wherein said one orplurality of detectably-labeled antibodies comprises at least oneantibody that is immunologically specific for one or a plurality ofbiological markers selected from c-kit, SCF, pAKT, and pc-kit, whereinsaid detectable label is a chromogen or a fluorophore;immunohistochemically staining a second breast tissue or second breastcell sample from the breast cancer patient with the one or the pluralityof detectably-labeled antibodies, wherein the detectable label is achromogen or a fluorophore, and wherein the second breast tissue or thesecond breast cell sample is taken from the breast cancer patient beforethe c-kit inhibitor has been administered; and determining an opticaldensity of the chromogen or fluorophore in the first tissue or firstcell sample and in the second tissue or second cell sample.
 2. Themethod of claim 1, wherein the c-kit inhibitor is imatinib mesilate(Gleevec™).
 3. The method of claim 1, wherein the one or the pluralityof biological markers is c-kit.
 4. The method of claim 1, wherein thec-kit inhibitor is imatinib mesilate (Gleevec™) and the one or theplurality of biological markers is c-kit.
 5. A method of treating apatient having a breast cancer, said method comprising:immunohistochemically staining a first breast tissue or first breastcell sample from a breast cancer patient to whom a c-kit inhibitor hasbeen administered with one or a plurality of detectably-labeledantibodies, wherein said one or plurality of detectably-labeledantibodies comprises at least one antibody that is immunologicallyspecific for one or a plurality of biological markers selected fromc-kit, SCF, pAKT, and pc-kit, wherein said detectable label is achromogen or a fluorophore; immunohistochemically staining a secondbreast tissue or second breast cell sample from the breast cancerpatient with the one or the plurality of detectably-labeled antibodies,wherein the detectable label is a chromogen or a fluorophore, andwherein the second breast tissue or the second breast cell sample istaken from the breast cancer patient before the c-kit inhibitor has beenadministered; and determining an optical density of the chromogen orfluorophore in the first tissue or first cell sample and in the secondtissue or second cell sample; and treating the breast cancer patientwith a c-kit inhibitor if decreased expression or activation of the oneor the plurality of biological markers is determined.
 6. The method ofclaim 5, wherein the c-kit inhibitor is imatinib mesilate (Gleevec™). 7.The method of claim 5, wherein the one or the plurality of biologicalmarkers is c-kit.
 8. The method of claim 5, wherein the c-kit inhibitoris imatinib mesilate (Gleevec™) and the one or the plurality ofbiological markers is c-kit.